Heterologous surface display of proteins (Stahl and Uhlen, TIETECH May 1997, 15 185-192) on recombinant microorganisms via the targeting and anchoring of heterologous proteins to the outer surface of host-cells such as yeast, fungi, mammalian and plant cells, and bacteria has been possible for several years. Display of heterologous proteins at these cells' surfaces has taken many forms, varying from the expression of reactive groups such as antigenic determinants, heterologous enzymes, (single-chain) antibodies, polyhistidyl tags, peptides, and other compounds. Heterologous surface display has been applied as a tool for research in microbiology, molecular biology, vaccinology, and biotechnology, and several patent applications have been filed.
Another application of bacterial surface display has been developing live-bacterial-vaccine delivery systems. The cell-surface display of heterologous antigenic determinants has been advantageous for inducing antigen-specific immune responses when using live recombinant cells for immunization. Another application has been the use of bacterial surface display in generating whole cell bioadsorbents or biofilters for environmental purposes, microbiocatalysts, and diagnostic tools.
In general, one has used chimeric proteins consisting of an anchoring or targeting part specific and selective for the recombinant organism used and has combined this part with a part comprising a reactive group as described above. A well known anchoring part, for example, comprises the so-called LPXTG box that binds covalently to a Staphylococcus bacterial surface, i.e., in the form of a fully integrated membrane protein. In this way, chimeric proteins are composed of at least two (poly)peptides of different genetic origin joined by a normal peptide bond. For example, in PCT International Publication Number WO 94/18830 relating to the isolation of compounds from complex mixtures and the preparation of immobilized ligands (bioadsorbents), a method has been claimed for obtaining such a ligand which comprises anchoring a binding protein in or at the exterior of the cell wall of a recombinant cell. The binding protein is essentially a chimeric protein produced by the recombinant cell, and is composed of an N-terminal part, derived from, for example, an antibody, that is capable of binding to a specific compound joined with a C-terminal anchoring part, derived from an anchoring protein purposely selected for being functional in the specific cell chosen. In PCT International Patent application Publication WO 97/08553 a method has been claimed for the targeting of proteins selectively to the cell wall of Staphylococcus spp, using as anchoring proteins long stretches of at least 80-90 amino acid long amino acid cell wall-targeting signals derived from the lysostaphin gene or amidase gene of Staphylococcus which encode for proteins that selectively bind to Staphylococcus cell wall components.
Vaccine delivery or immunization via attenuated bacterial vector strains expressing distinct antigenic determinants against a wide variety of diseases is now commonly being developed. Recently, mucosal (for example nasal or oral) vaccination using such vectors has received a great deal of attention. For example, both systemic and mucosal antibody responses against an antigenic determinant of the hornet venom were detected in mice orally colonized with a genetically engineered human oral commensal Streptococcus gordonii expressing the antigenic determinant on its surface (Medaglini et al., PNAS 1995, 2; 6868-6872). Also, a protective immune response could be elicited by oral delivery of a recombinant bacterial vaccine wherein tetanus toxin fragment C was expressed constitutively in Lactococcus lactis (Robinson et al., Nature Biotechnology 1997, 15; 653-657). Especially mucosal immunization as a means of inducing IgG and secretory IgA antibodies directed against specific pathogens of mucosal surfaces is considered an effective route of vaccination. Immunogens expressed by bacterial vectors are presented in particulate form to the antigen-presenting cells (for example M-cells) of the immune system and should, therefore, be less likely to induce tolerance than soluble antigens. In addition, the existence of a common mucosal immune system permits immunization on one specific mucosal surface to induce secretion of antigen-specific IgA, and other specific immune responses at distant mucosal sites. A drawback to this approach is the potential of the bacterial strain to cause inflammation and disease in itself, potentially leading to fever and bacteremia. An alternative approach avoids the use of attenuated bacterial strains that may become pathogenic themselves by choosing recombinant commensal bacteria as vaccine carriers, such as Streptococcus spp. and Lactococcus spp.
However, a potential problem with such recombinant organisms is that they may colonize the mucosal surfaces, thereby generating a long-term exposure to the target antigens expressed and released by these recombinant microorganisms. Such long term exposure can cause immune tolerance. In addition, the mere fact alone that such organisms are genetically modified and contain recombinant nucleic acid is meeting considerable opposition from the lay public as a whole, stemming from a low level of general acceptance for products containing recombinant DNA or RNA. Similar objections exist against the use of even-attenuated strains of a pathogenic nature or against proteins or parts of proteins derived from pathogenic strains. However, as explained above, present techniques of heterologous surface display of proteins in general entail the use of anchoring or targeting proteins that are specific and selective for a limited set of microorganisms which in general are of recombinant or pathogenic nature, thereby greatly restricting their potential applications.